Project Summary Defects in many known regulators of lumenal Ca2+ in lysosomes lead to distinct neurological disorders by disrupting lysosomal Ca2+ levels. Lysosomal Ca2+ can be dysregulated by defects in either Ca2+ release channels or transporters. While there are several examples of lysosomal Ca2+ release channels, there is only one example of a protein that facilitates lysosomal Ca2+ import. This solitary example was identified by my lab to be catp-6 in C. elegans. Its human homolog, ATP13A2, is one of the major risk genes for Parkinson's disease (PD). Given that the only example of a protein that drives lysosomal Ca2+ import is a risk gene for PD, an understanding of its function, as well as identifying more regulators of lysosomal Ca2+ entry is of high significance. Catp-6 was identified as a potential lysosomal Ca2+ importer only because of a new fluorescent reporter called CalipHluor, with which one could quantitatively image lysosomal Ca2+. Here we propose to identify and catalog proteins that drive lysosomal Ca2+ import by screening nematodes that are engineered to show phenotypes at the whole worm, sub-cellular and lysosomal Ca2+ level. We have developed a system where, the deletion of a lysosomal Ca2+ importer will concurrently rescue lethality caused by the deletion of a lysosomal Ca2+ release channel, restore lysosome size and restore lysosomal Ca2+ - the latter revealed by Ca2+ mapping with CalipHluor. Since our methodology can map defects in lysosomal Ca2+ uptake, we propose to investigate two new molecular mechanisms that drive lysosomal Ca2+ import. The first is a gene we denote LCAX-1, that we identified from a preliminary screen in nematodes, that has putative Ca2+/H+ exchanger (CAX)-like function. While CAX genes have been described in diverse organisms, lysosomal CAX genes have eluded discovery in humans. We propose experiments that will test whether LCAX-1 behaves like a CAX at the lysosome and if so, it would constitute the first example of a human lysosomal CAX. The second corresponds to the Parkinson's risk gene ATP13A2 that has been previously described as an importer of various heavy metal ions. Yet our data suggest that it imports Ca2+. We propose experiments that will distinguish between these two possibilities and by mutational analysis we will identify the residues in ATP13A2 responsible for Ca2+ binding. Success in our aims will offer knowledge of new lysosomal Ca2+ import mechanisms, identify the elusive human lysosomal CAX gene, and provide structural insight into the functionality of a risk gene for Parkinson's disease e.g., identify the Ca2+ binding site in ATP13A2.